1. Field of the Invention
The present invention is directed to a method of producing a vaccine effective against one or more Escherichia coli (E. coli) strains. The present invention futher relates to a polyvalent vaccine composed of nonpyrogenic, nontoxic, immunogenic serotype-specific LPS based conjugates and to the conjugates therein.
2. Description of the Art
Escherichia coli (E. coli) is the leading cause of life-threatening gram-negative bacterial sepsis. Both capsular (K) and lipopolysaccharide (LPS) (O) antigens are important virulence factors as described in Cross, A. S., Kim, K. S., Wright, D. C., Sadoff, J. C., Gemski, P. , "Role of lipopolysaccharide and capsule in the serum resistance of bacteremic strains of Escherichia coli," J. Infect. Dis. 154: 497-503, 1986; and Pluschke, G., Mayden, J., Achtman, M., Levine, R. P., "Role of the capsule and O antigen in resistance of O18:K1 Escherichia coli to complement-mediated killing," Infect. Immun. 42: 907-913, 1983. Both capsular and LPS antigens can confer protection against the bactericidal effect of normal human serum, a characteristic which allows E. coli to invade and persist in the bloodstream as noted in Cross, A. S., Kim, K. S., Wright, D. C., Sadoff, J. C., Gemski, P., "Role of lipopolysaccharide and capsule in the serum resistance of bacteremic strains of Escherichia coli," J Infect. Dis. 154: 497-503, 1986; and Pluschke, G., Mayden, J., Achtman, M., Levine, R. P., "Role of the capsule and O antigen in resistance of O18:K1 Escherichia coli to complement-mediated killing," Infect. Immun. 42: 907-913, 1983.
Serospecific antibodies directed against either the capsular or LPS antigen can afford protection against experimental E. coli infections in animals as described by Cross, A. S., Zollinger, W., Mandrell, R., Gemski, P., Sadoff, J. C., "Evaluation of immunotherapeutic approaches for the potential treatment of infections caused by K1-positive Escherichia coli," J Infect. Dis. 197: 68-76, 1983; and Kaijser, B., Ahlstedt, S., "Protective capacity of antibodies against Escherichia coli O and K antigens," Infect. Immun. 17: 286-289, 1977. A limited number of both O and K antigens are expressed by E. coli strains which cause serious infections, such as septicemia, making vaccines composed of either antigen feasible as noted in Orskov, F, Oskov, I., "Escherichia coli extraintestinal infections," J. Hyg 95: 551-575, 1985; Cross, A. S., Gemski, P., Sadoff, J. C., Orskov, F., Oskov, I., "The importance of the K1 capsule in invasive infections caused by Escherichia coli," J. Infect. Dis. 149: 184-193, 1984; and McCabe, W. R., Kaijser, B., Olling, S., Uwaydah, M., Hanson, L. A., "Escherichia coli in bacteremia: K and O antigens and serum sensitivity of strains from adults and neonates," J. Infect. Dis. 138: 33-41, 1978.
There are, however, two major drawbacks to the use of E. coli capsular antigens as human vaccines. First approximately 40% of E. coli bacteremic isolates cannot be serotyped as relates to capsular antigen. In addition, the K1 and K5 capsular antigens, which are expressed by more than 20% of K-typeable blood isolates, are poorly immunogenic in humans due to their antigenic cross-reactivity with mammalian glycosaminoglycans. Therefore, a K antigen-based E. coli vaccine would have a limited coverage, and hence, little utility.
Based upon the above finding, a serospecific LPS-based vaccine would appear to possess the greatest potential to protect against E. coli extraintestinal infections. Native LPS, however, is far too toxic and pyrogenic for use as a human vaccine. The O serospecificity of E. coli is contained within the O-polysaccharide (O-PS) moiety of the LPS molecule as for other gram-negative bacteria. The O-PS region can be separated from the toxic lipid A portion of the LPS molecule by cleavage in dilute acetic acid followed by pelleting of the insoluble lipid A moiety by centrifugation. While O-PS isolated in this manner is serologically reactive nontoxic and non pyrogenic, it is non-immunogenic due to its small molecular weight as noted in Pier, G. B., Sidberry, H. F., Sadoff, J. C., "Protective immunity induced in mice by immunization with high molecular polysaccharide from Pseudomonas aeruginosa," Infect. Immun. 22: 919-925, 1978; and Chester, I. R., Meadow, P. M., Pitt, T. L., "The relationship between O-antigenic lipopolysaccharides and serological specificity in strains of Pseudomonas aeruginosa of different O-serotypes," J. General Microbiol. 78: 305-318, 1973.
One means by which to produce a protective immune response to isolated O-PS is to covalently couple it to a carrier protein, yielding a conjugate vaccine. Escherichia coli O18 O-PS has been covalently coupled to both cholera toxin and Pseudomonas aeruginosa toxin A, yielding safe, immunogenic, and protective monovalent conjugate vaccines as described in Cryz, S. J., Jr., Cross, A. S., Sadoff, J. C., Furer, E., "Synthesis and characterization of Escherichia coli O18 )-polysaccharide conjugate vaccines," Infect. Immun. 58: 373-377, 1990; and Cryz, S. J., Jr., Cross, A. S., Sadoff, J. C., Wegmann, A., Que, J. U., Furer, E., "Safety and immunogenicity of Escherichia coli O18 O-specific polysaccharide (O-PS)-toxin A and O-PS-cholera toxin conjugate vaccines in humans," J. Infect. Dis. 163: 1040-1045, 1991.
However, any vaccine against E. coli based on serospecific O-PS determinants would have to be multi-valent based upon the observation that the majority (.about.70%) of bacteremic infections are caused by 10-12 different serotypes of E. coli. Serospecificity is conferred by both the monosaccharide composition of O-PS and the type of chemical linkage between each monosaccharide as noted in Orskov, F., Orskov, I., "Serotyping of Escherichia coli," Methods in Microbiology 14: 43-112, 1984. Therefore, the conditions used to synthesize the above described O18 monovalent conjugate might not be suitable for other serotypes of E. coli.
By the present invention, isolated O-PS from 12 serotypes of E. coli were covalently coupled to P. aeruginosa toxin A which serves as a "carrier protein" for the O-PS. The conditions employed to covalently couple toxin A to E. coli O-PS effectively detoxify the toxin A molecule and preserve the antigenicity of the O-PS moiety. The resulting polyvalent conjugate was found to be safe and immunogenic in humans when administered by the parenteral route.